Directed protein evolution is a powerful technology for generating proteins with desired properties, such as thermostability. There is currently no effective in vitro method to select proteins with enhanced thermostability. Thermostable proteins have a wide range of industrial and medical applications. The aim of this project is to construct a reconstituted in vitro protein synthesis system (thermoPURE system) using purified components from Thermus thermophilus, a bacterium that grows at an optimal temperature of 72oC. The thermoPURE system may allow protein synthesis to be conducted in vitro at elevated temperatures, and due to its low background activities, facilitate in vitro screening and selection or other applications that require elevated temperatures. In conjunction with powerful in vitro selection technologies such as in vitro compartmentalization and ribosome display, the thermoPURE may become the only system that allows in vitro directed evolution of thermostable proteins from their mesophilic origins. During the initial phase of the project, we will clone a complete set of genes for the translation factors, aminoacyl-tRNA synthetases and energy regeneration enzymes from Thermus thermophilus. All proteins will be overexpressed and purified from E. coli. The ribosomes and tRNAs will be purified from a growing culture of Thermus thermophilus. Using our experience and capacity of manufacturing the E. coli version of the reconstituted protein synthesis system (the PURE system), we will reconstitute the protein translation machinery of Thermus thermophilus and demonstrate in vitro protein synthesis at elevated temperatures. In the second phase, we will apply the thermoPURE system for in vitro evolution of mesophilic proteins such as restriction enzymes, RNA polymerases, and single-chain antibodies with an ultimate goal to generate valuable proteins with enhanced thermostability for industrial and medical usage. PUBLIC HEALTH RELEVANCE: Proteins that are stable at elevated temperatures have a wide range of industrial and medical applications. There is currently no effective in vitro method to select proteins with enhanced thermostability. The goal of this project is to provide such a method by constructing a reconstituted in vitro protein synthesis system from purified components of Thermus thermophilus, a bacterium that grows at an optimal temperature of 72oC.